Mss4 as an antifungal target

ABSTRACT

The invention provides 1-phosphotidylinositol-4-phosphate 5-kinase (MSS4) as a novel antifungal target, screening methods for MSS4 inhibitors and their use as antifungal compounds, pharmaceutical compositions containing them and their use in medicine.

The present invention relates to a novel antifugal target,1-phosphotidylinositol-4-phosphate 5-kinase (MSS4), screening methodsfor MSS4 inhibitors and their use as antifungal compounds,pharmaceutical compositions containing them and their use in medicine,specifically in the treatment of an individual susceptible to orsuffering from an anti-fungal infection. In particular the compoundsfind use in the treatment of topical or mucosal (e.g. thrush and vaginalcandidiasis) fungal infections, e.g. caused by fungus of the Candidaspecies, and for systemic infections, e.g. caused by fungi of Candidaand Aspergillus species, such as but not limited to C. albicans,Aspergillus flavus or Aspergillus fumigatus.

INTRODUCTION

Fungal Pathogens

Two major fungal pathogens are those of the Candida species, such as butnot limited to, C. albicans, and those of the Aspergillus species, suchas but not limited to, Aspergillus flavus or Aspergillus fumigatus.

Fungal infections can affect humans and animals. Generally, fungalinfections occur as a result of opportunistic infection of a weakened orimmune-suppressed individual and these can include infections of thejoints and skin. The yeast Candida albicans (C. albicans) is one of themost pervasive fungal pathogens in humans. It is the cause of anincreasing financial and logistic burden on the medical care system andits providers. Although C. albicans is a member of the normal flora ofthe mucous membranes in the respiratory, gastrointestinal, and femalegenital tracts, it may gain dominance in such locations (e.g. upontreatment with antibacterial antibiotics, in patients with diabetes orin patients using corticosteroids) and be associated with pathologicconditions. In addition, almost all HIV-positive individuals suffer froma Candida infection prior to the onset of developing full-blown ADS. Theincidence of life-threatening fungal infections has increaseddramatically as the population of immunocompromised individuals.(including cancer, organ transplant and AIDS patients) has increased.Present therapeutic options for the treatment of these infections arelimited and thus there is a need for new anti-fungal compounds withnovel mechanisms of action for use in treating or preventing such fungalinfections.

Antifungal drug development often relies on the screening of a largenumber of compounds before one or more lead compounds are found that areeffective against the target fungi. Thus, it is critical for thedevelopment of these screens to define proteins essential for survivalor growth of the target fungi and to discover means of purifying orproducing such proteins. Thus, there is a need in the art to identifyessential fungal structural or functional gene products that can serveas targets for drug intervention, and for methods for identifying usefulanti-fungal agents that impair the function of these essential fungalgene products, and for compositions that can be used to treat fungalinfections by preventing or inhibiting the growth of, and preferentiallykilling, the fungi.

Identification of “Essential” Genes

Varying definitions are used in the art for what constitutes anessential gene, but the term is most frequently applied to those genesnecessary for growth on rich media This variation in the art can bemisleading and restrictive in terms of identifying gene products thatconstitute good antifungal targets. A significant amount of C. albicansgenomic sequence information is available in both public(http://www.sequence.stanford.edu/group/candida/) and private (IncyteGenomics Inc.) databases. This can be combined with genomic sequencedata from other organisms (The yeast genome directory, 1997, Nature,387(6632 Suppl):5; Wood V, et al, 2002, Nature, 415(6874):871-80) andwith supporting data such as the functional profiling of theSaccharomyces cerevisiae genome (Giaever G, et al, 2002, Nature,418(6896):387-91). This bioinformatics driven approach has allowed theprediction of genes that may be essential in C. albicans (Spaltmann F,et al, 1999, Drug Discovery Today, 4:17-26). However, even forrelatively closely related organisms such as Saccharomyces cerevisiaeand C. albicans, there are significant differences that make such insilico predictions unreliable. For example, CET1 and CDC25 are notessential in C. albicans despite being essential in Saccharomycescerevisiae (Enloe B, et al, 2000, J. Bacteriol., October, 182:20,5730-6; Dunyak D S, et al, 2002, 6^(th) ASM Conference on Candida andCandidiasis).

There are several strategies for identifying essential genes in C.albicans by practical methodology. Negative approaches rely on theinability to generate a strain that contains a disrupted functionaltarget gene. The majority of genes characterised in this way rely onvariations of the URA blaster method (Fonzi W A & Irwin M Y, 1993,Genetics, 134:717-728). These techniques can be highly effective foranalysing individual genes, but they may not be completely reliable.CET1 was incorrectly reported to be essential in C. albicans becauseviable homozygous mutants could not be recovered using the URA blastermethod (Pei, et al, 2001). However it has subsequently been shown not tobe essential (Dunyak, et at, 2002). Positive approaches control theexpression of the target gene either indirectly, such as using antisenseRNA (De Backer M D, et al, 2001, Nat. Biotechnol., March, 19:3, 235-41),or directly such as promoter replacement with inducible promoters suchas MRP1 and Tet (Munro C A, et al, 2001, Mol. Microbiol., March 39:51414-26; Nakayama H, et al, 2000, Infect. Immun., December 68:126712-9).

Genome wide identification of essential genes has not been successfullyapplied to C. albicans for several reasons. These include that C.albicans is a diploid organism, is not capable of mating under normalcircumstances, and that there are few functional transposable elements.Attempts to overcome these issues by using antisense RNA and promoterinterference have had limited success (De Backer, et al, 2001).Therefore there is a need in the art for validated essential genes offungal species, in particular the Candida species, that can be used astargets for the development of new antifungal compounds.

1-Phosphotidylinositol-4-phosphate 5-kinase

1-Phosphotidylinositol-4-phosphate 5-kinase (MSS4) E.C. 2.7.1.68 isinvolved in inositol phosphate metabolism and functions to convertATP+1-phosphotidyl-1D-myo-inositol4-phosphate→ADP+1-phosphotidyl-1D-myo-inositol 4,5-bisphosphate (PIP2)(Yoshida et al, 1994, Mol. Gen. Genet. 242, 631-640; Hairfield et al,2002, Microbiology, 148(6), 1737-1746). The MSS4 enzyme is encoded bythe MSS4 gene (YDR208W/YD8142A.05) and details for the fungal enzyme areprovided under Accession numbers: P38994 in the Swiss Prot database(http://ca.expasy.org) for S. cerevisiase; CA0623 in the InstitutPasteur Candida database (http://genolist.pasteur.fr/CandidaDB/) whichis cross-referenced with the Stanford open-reading frame (ORF) orf6.1660(http://www.sequence.stanford.edu/group/candida/). Synonyms for MSS4include diphosphoinositide kinase, PIP kinase (PIP5K),PtdIns(4)P-5-kinase and CaMSS4.3.

Homma K, et al, 1998, J. Biol, Chem., 273:25, 15779-15786 andDesrivieres S, et al, 1998, J. Biol. Chem., 273:25, 15787-15793 describethe role of MSS4 in actin organisation in S. cerevisiase.

The present invention is based on the finding that MSS4 is an essentialprotein for the fungal species Candida and Aspergillus. This findingdemonstrates the potential for developing fungal selective MSS4inhibitors, which can kill invading fungal organisms while sparing thehost of any detrimental effects. Prior to this invention, MSS4 has notbeen considered as a differential target for antifungal compounds.

SUMMARY OF THE INVENTION

The present invention relates to fungal1-phosphotidylinositol-4-phosphate 5-kinase (hereinafter referred to as“MSS4”) as a target for antifungal therapy, in particular, forantifungal therapy against Candida and Aspergillus species. Theinvention also relates to a method for screening or testing forpotential antifungal compounds, e.g. small molecules, by determiningwhether a candidate agent is capable of specifically inhibiting fungalphosphorylation activity via a selective interaction with MSS4. Thepresent invention describes the essential nature of MSS4 in C. albicans.It further describes the use of mechanism-based assays, with or withoutthe use of a transformed eukaryotic organism with the MSS4 gene underthe control of a heterologous promoter, to facilitate drug discovery.

Additionally, the invention relates to MSS4 inhibitor compositions andto methods for treating fungal infections, e.g. Candida and Aspergillusfungal infections, by administering to a host suffering from a fungalinfection a therapeutically effective amount of a MSS4 inhibitor.

Definitions

In the context of this invention:

“Essential gene” is defined as a fungal gene necessary for growth onrich medium.

“MSS4 inhibitor” is defined as any compound that impairs MSS4 functionin the fungus. A compound that impairs MSS4 function may be one that,modulates, e.g. inhibits, the expression or activity of MSS4, interactswith MSS4 or binds to MSS4. Furthermore, a compound that modulates theexpression of MSS4 may interfere with the transcription of the geneencoding MSS4 or with the translation of mRNA encoding MSS4 in targetorganisms. It is desirable that the compound shows specificity forfungal over host MSS4. A therapeutically effective amount of a MSS4inhibitor is one that is sufficient to inhibit partially or fully thephosphorylation activity via MSS4 of the causative fungi.

“Fragment” is defined as a fragment of a MSS4 polypeptide e.g. asprovided by accession numbers P38994, CA0623, or Stanford orf6.1660,having at least 70%, more preferably it has at least 75%, at least 80%,at least 85%, at least 90%, at least 95% or at least 98% identity to thenative polypeptide over the length of the fragment and which is at leastten amino acids long. An active fragment is one that retains the abilityto carry out the MSS4 enzyme function.

“Function-conservative fragment” is defined as a MSS4 encoding sequencein which a given amino acid residue in the polypeptide has been changedwithout altering the overall conformation and function of the nativepolypeptide, including, but not limited to, replacement of an amino acidwith one having similar physical and/or chemical properties (such as,for example, acidic, basic, hydrophobic, and the like) or polymorphisms.

“Fusion protein” unless otherwise specified, is defined as a MSS4polypeptide, fragment or function-conservative fragment thereof fusedvia a covalent bond (e.g. a peptide bond), at optionally the N-terminusor the C-terminus, to an amino acid sequence of another protein (orportion thereof; preferably at least a 10, 20 or 50 amino acid portionof the protein). Preferably the polypeptide, or fragment thereof, islinked to the other protein at the N-terminus of the constant domain ofthe polypeptide.

“Growth” is defined as the normal growth pattern of fungi, i.e. the celldoubling time during the log phase of growth. For example, in richmedia, wild-type C. albicans has a doubling time of approximately 60minutes. Growth of the cells may be measured by following the opticaldensity of cells in liquid media, where an increasing optical densityindicates growth. Alternatively, growth can also be measured by colonyformation from single cells on solid media plates.

“Viability” is defined as the ability of fungal cells to resume growthfollowing a treatment of the cells that results in cessation of growthOne typical means by which viability is measured is by testing theability of cells to form colonies on solid media plates.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides MSS4 as a specific target for antifungalcompounds.

The methods of the invention provide a facile and specific assay toscreen compounds as potential antifungal compounds, in particular, asantifungal compounds against Candida and Aspergillus species.

Thus, the invention provides a method of screening or testing forantifungal compounds, e.g. against Candida or Aspergillus species, thatimpair 1-phosphotidylinositol-4-phosphate 5-kinase enzyme function(MSS4), comprising:

-   -   a) providing fungal MSS4, preferably Candida or Aspergillus        MSS4;    -   b) providing one or more candidate compounds;    -   c) contacting said MSS4 with said one or more candidate        compounds; and    -   d) determining the interaction of the candidate compound with        said MSS4.

The screening method of the invention may be performed using techniquesknow in the art, e.g. the assay may comprise a growth inhibition assay,a binding assay or a translation inhibition assay. Binding assaysinclude competitive binding assays, wherein the binding affinity of thecandidate compound is compared with that of a known enzyme substrate forMSS4, a preferred enzyme substrate is adenosine triphosphate (ATP).

In the screening methods of the invention the candidate compound orenzyme substrate may be labelled to allow easy quantitation of theinteraction between the candidate compound and the enzyme. Preferablythe substrate is labelled e.g. using a radiolabel such as but notlimited to, ³³P and the preferred substrate is adenosine triphosphate(ATP), as described in Example 3.

MSS4 may be cloned or purified from fungi for use in in vitro binding,ligand binding or translation inhibition assays. Preferably, the MSS4 isfrom fungal pathogens of humans and animals, such as Candida orAspergillus species. In a particular embodiment, MSS4 may comprise afragment, a function-conservative variant, an active fragment or afusion protein of MSS4.

MSS4 can be purified by techniques well known to those skilled in theart. Methods for polypeptide purification include, without limitation,preparative disc-gel electrophoresis, isoelectric focusing, HPLC,reversed-phase HPLC, gel filtration, ion exchange and partitionchromatography, and countercurrent distribution. For some purposes, itis preferable to produce the polypeptide in a recombinant system inwhich the protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence. Thepolypeptide can then be purified from a crude lysate of the host cell bychromatography on an appropriate solid-phase matrix. Alternatively,antibodies produced against the fungal target protein or againstpeptides derived therefrom can be used as purification reagents.

MSS4 can also be provided in a transformed eukaryotic organism under thecontrol of a heterologous promoter. Such cells can be used in growthinhibition assays. Preferably, the eukaryotic organism is C. albicans orS. cervisiae. More preferably the organism is C. albicans. Briefly, a C.albicans strain is generated in which expression of the MSS4 gene can betightly regulated. To do this the wild-type allele of the gene ofinterest is replaced with an allele that can be regulated by anexogenous agent. In general, nucleic acid manipulations and otherrelated techniques used in practicing the present invention employmethods that are well known in the art, as disclosed in, e.g. MolecularCloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis,Cold Spring Harbor) and Current Protocols in Molecular Biology (Eds.Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ.Assoc., Wiley Interscience, NY, N.Y., 1997).

Thus, the invention also provides a modified eukaryotic cell(s) whereinthe cell(s) expresses MSS4 under the control of a heterologous promoter.In one embodiment, the MSS4 may be heterologous or homologous.Preferably, the MSS4 is homologous.

The eukaryotic cell is preferably C. albicans or S. cervisiae, morepreferably C. albicans.

In a specific embodiment, MSS4 may be expressed in atetracycline-regulatable expression system (as described in Example 4).The tetracycline-regulatable expression system is an established toolfor conditional expression of eukaryotic genes (Gossen M A, & H Bujard,1992, Proc. Natl. Acad. Sci. USA, 89:5547-5551; Nagahashi S, et al,1997, Mol. Gen. Genet, 255:372-375; Nakayama H, et al, 1998,Microbiology, 144:2407-2415; Nakayama H, et al, 2000, Infect. Immun.,Dec 68:12 6712-9). Such a system consists of two components derived fromthe Tn10 transposon of Escherichia coli (Hillen W, & A Wissmann, 1989,In Protein-nucleic acid interaction, vol. 10, Macmillan Press, London,United Kingdom, p. 143-162). The first component comprises a minimalpromoter element downstream of a tetracycline operator sequence (tetO),which replaces the natural promoter of the target gene. The secondcomponent is a transactivator, that is a fusion protein comprising atranscriptional activation domain and the tetracycline repressor protein(TetR).

In the absence of tetracycline, TetR specifically binds to tetO as adimer, resulting in the activation of transcription by recruiting thetransactivator to the promoter. When tetracycline is present, it bindsto the TetR repressor with high affinity and inhibits dimerisation,thereby preventing binding to tetO. Therefore, MSS4 gene expression isactivated in the absence, and repressed in the presence, oftetracycline. A synthetic tetracycline derivative, such as but notlimited to, doxycycline can be used to control the expression of theMSS4 gene as described above.

The tetracycline-regulatable expression system has several advantagesover alternative systems. It was derived from a prokaryotic system so itis not anticipated to show pleiotropic effects (Gossen and Bujard,1992), it can be used in an animal host (Nakayama H, et al, 1998,Microbiology, 144:2407-2415; Nakayama H, et al, 2000, Infect. Immun.,Dec 68:12 6712-9), it is highly specific, and non-toxic.

Such modified cells may be used in screening methods. Thus, theinvention also provides a method of screening or testing for candidateanti-fungal compounds, e.g. against Candida or Aspergillus species, thatimpair 1-phosphotidylinositol-4-phosphate 5-kinase enzyme (MSS4)function, comprising;

-   -   a) providing fungal MSS4, preferably Candida or Aspergillus        MSS4, in a eukaryotic cell(s) that expresses MSS4 under the        control of a heterologous promoter;    -   b) providing one or more candidate compounds;    -   c) contacting said eukaryotic cell(s) with said one or more        candidate compounds; and    -   d) determining the interaction of the candidate compound with        said MSS4 by assessing the effect on growth or viability of said        cells.

The screening methods of the invention include both in vitro and in vivomethods. Candidate compounds which may be screened according to themethods of the invention include small molecules and peptides. Thecandidate compounds may be synthetic compounds, a mixture of syntheticcompounds, a crude preparation, a purified preparation or an initialextract of a natural product obtained from plant, microorganism oranimal sources.

The invention also provides a compound identified by the screeningmethods described above, which impairs MSS4 function and is referred toherein as a “MSS4 inhibitor”.

MSS4 inhibitors of the invention are useful as antifungal compounds.Thus, they may be used in the treatment and prevention of various fungalinfections such as topical or mucosal (e.g. thrush and vaginalcandidiasis) fungal infections, caused by e.g. Candida species, and forsystemic fungal infections, caused by e.g. Candida and Aspergillusspecies, such as but not limited to C. albicans, Aspergillus flavus orAspergillus fumigatus.

For the purposes of this invention, the medicament can be used in thecurative or prophylatic treatment of fungal infections in humans andanimals, especially domestic animals such as dogs, cats, horses etc.

In addition, the MSS4 inhibitors also find use in the curative orprophylatic treatment of fungal infections in subjects who areimmunosuppressed e.g. as a result of a therapy (e.g. chemotherapy orradiotherapy), organ transplant or an infection (e.g. HIV).

In additional embodiments, therefore, the present invention provides:

-   -   i) the use of a MSS4 inhibitor as an anti-fungal agent.    -   ii) the use of a MSS4 inhibitor in the manufacture of a        medicament for the treatment of fungal infections, such as        topical or mucosal (e.g. thrush and vaginal candidiasis) fungal        infections, e.g. caused by Candida species, and for systemic        fungal infections e.g. caused by Candida and Aspergillus        species, such as but not limited to, C. albicans, Aspergillus        flavus or Aspergillus fumigatus.    -   iii) the use of a MSS4 inhibitor in the manufacture of a        medicament for the treatment of fungal infections in a subject        who is immunosuppressed, for example, as a result of a therapy        (e.g. chemotherapy or radiotherapy), organ transplant or an        infection (e.g. HIV).    -   iv) a method for the treatment or prevention of fungal        infections in a host, such as topical or mucosal (e.g. thrush        and vaginal candidiasis) fungal infections, e.g. caused by        Candida species, and for systemic fungal infections, e.g. caused        by Candida and Aspergillus species, such as but not limited        to C. albicans, Aspergillus flavus or Aspergillus fumigatus,        which comprises administering to the host a therapeutically or        prophylactically effective amount of a MSS4 inhibitor.    -   v) a method for the treatment or prevention of fungal infections        in a subject who is immunosuppressed, for example, as a result        of a therapy (e.g. chemotherapy or radiotherapy), organ        transplant or an infection (e.g. HI)M which comprises the step        of administering to the subject a therapeutically or        prophylactically effective amount of a MSS4 inhibitor.

In order to use MSS4 inhibitors in therapy (human or veterinary), theywill normally be formulated into a pharmaceutical composition inaccordance with standard pharmaceutical practice, e.g. by admixing theMSS4 inhibitor and a pharmaceutically acceptable carrier.

Thus according to a further aspect of the invention there is provided apharmaceutical composition comprising a MSS4 inhibitor and apharmaceutically acceptable carrier. The pharmaceutical compositions areparticularly useful in the prevention or treatment of fungal infections,preferably, in the treatment of Candida or Aspergillus fungalinfections.

MSS4 inhibitors may be administered to a host by any of the routesconventionally used for drug administration, for example they may beadministered parenterally, orally, topically (including buccal,sublingual or transdermal) or by inhalation. The most suitable route foradministration in any given case will depend on the particular MSS4inhibitor, the infectious organism involved, the host, and the natureand severity of the disease and the physical condition of the host.

The MSS4 inhibitors may be administered in combination, e.g.simultaneously, sequentially or separately, with one or more othertherapeutically active, e.g. antifungal, compounds.

The dosage to be administered of a MSS4 inhibitor will vary according tothe particular MSS4 inhibitor, the infectious organism involved, thehost, the severity of the disease, physical condition of the host, andthe selected route of administration; the appropriate dosage can bereadily determined by a person skilled in the art. For the treatment offungal diseases in humans and animals, the dosage may range from 0.01mg/kg to 750 mg/kg. For prophylactic use in human and animals, thedosage may range from 0.01 mg/kg to 100 mg/kg.

The compositions may contain from 0.1% by weight, preferably from 10-60%by weight, of the MSS4 inhibitor, depending on the method ofadministration.

Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of MSS4 inhibitor per dose. Sucha unit may contain for example but without limitation, 100 mg/kg to 0.1mg/kg depending on the condition being treated, the route ofadministration and the age, weight and condition of the host. Preferredunit dosage compositions are those containing a daily dose or sub-dose,as recited above, or an appropriate fraction thereof, of the activeingredient

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an agent of the inventionwill be determined by the nature and extent of the condition beingtreated, the form, route and site of administration, and the particularhost being treated, and that such optimums can be determined byconventional techniques. It will also be appreciated by one of skill inthe art that the optimal course of treatment, i.e. the number of dosesof an agent of the invention given per day for a defined number of days,can be ascertained by those skilled in the art using conventional courseof treatment determination tests.

Dosage regimens are adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

Pharmaceutically acceptable carriers for use in the invention may take awide variety of forms depending, e.g. on the route of administration.

Compositions for oral administration may be liquid or solid. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Oral liquid preparations may containsuspending agents, for example sorbitol, methyl cellulose, glucosesyrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose,aluminium stearate gel or hydrogenated edible fats, emulsifying agents,for example lecithin, sorbitan monooleate, or acacia; water; non-aqueousvehicles (which may include edible oils), for example almond oil, oilyesters such as glycerine, propylene glycol, or ethyl alcohol;preservatives, for example methyl or propyl p-hydroxybenzoate or sorbicacid; flavoring agents, preservatives, coloring agents and the like maybe used.

In the case of oral solid preparations such as powders, capsules andtablets, carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegratingagents, and the like may be included. Because of their ease ofadministration, tablets and capsules represent the most advantageousoral dosage unit form in which case solid pharmaceutical carriers aregenerally employed. In addition to the common dosage forms set outabove, MSS4 inhibitors may also be administered by controlled releasemeans and/or delivery devices. Tablets and capsules may compriseconventional carriers or excipients such as binding agents for example,syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone;fillers, for example lactose, sugar, maize-starch, calcium phosphate,sorbitol or glycine; tableting lubricants, for example magnesiumstearate, talc, polyethylene glycol or silica; disintegrants, forexample potato starch; or acceptable wetting agents such as sodiumlauryl sulphate. The tablets may be coated by standard aqueous ornon-aqueous techniques according to methods well known in normalpharmaceutical practice.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient, as a powder or granules, or as a solution or a suspension inan aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or awater-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association the active ingredient with the carrier, whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. For example, a tablet may be prepared by compression ormoulding, optionally with one or more accessory ingredients.

Compressed tablets may be prepared by compressing, in a suitablemachine, the active ingredient in a free-flowing form such as a powderor granules, optionally mixed with a binder, lubricant, inert diluent,surface active or dispersing agent. Moulded tablets may be made bymoulding, in a suitable machine, a mixture of the powdered compoundmoistened with an inert liquid diluent. Desirably, each tablet containsfrom about 1 mg to about 500 mg of the active ingredient and each cachetor capsule contains from about 1 to about 500 mg of the activeingredient

Compositions comprising a MSS4 inhibitor may also be prepared in powderor liquid concentrate form. Conventional water soluble excipients, suchas lactose or sucrose, may be incorporated in the powders to improvetheir physical properties. Thus, particularly suitable powders of thisinvention comprise 50 to 100% w/w, and preferably 60 to 80% w/w of thecombination and 0 to 50% w/w and preferably 20 to 40% w/w ofconventional excipients. When used in a veterinary setting such powdersmay be added to animal feedstuffs, for example by way of an intermediatepremix, or diluted in animal drinking water.

Liquid concentrates of this invention for oral administration suitablycontain a water-soluble compound combination and may optionally includea pharmaceutically acceptable water miscible solvent, for examplepolyethylene glycol, propylene glycol, glycerol, glycerol formal or sucha solvent mixed with up to 30% v/v of ethanol.

Pharmaceutical compositions suitable for parenteral administration maybe prepared as solutions or suspensions of the MSS4 inhibitors in watersuitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene,glycols, and mixtures thereof in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include aqueous ornon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the composition isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. Extemporaneous injection solutions, dispersions and suspensionsmay be prepared from sterile powders, granules and tablets.

The compositions may be presented in unit-dose or multi-dose containers,for example in sealed ampoules and vials and to enhance stability, maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. The sterile liquid carrier may besupplied in a separate vial or ampoule and can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (e.g.glycerol, propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, and vegetable oils. Advantageously, agents such as alocal anaesthetic, preservative and buffering agents can be included thesterile liquid carrier.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols oroils, transdermal devices, dusting powders, and the like. Thesecompositions may be prepared via conventional methods containing theactive ingredient. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollients in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers may be present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or cream.When formulated in an ointment, the active ingredient may be employedwith either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for topical administration to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent. Theyalso include topical ointments or creams as above.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter or other glycerideor materials commonly used in the art, and the suppositories may beconveniently formed by admixture of the combination with the softened ormelted carrier(s) followed by chilling and shaping moulds. They may alsobe administered as enemas.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraycompositions. These may comprise emollients or bases as commonly used inthe art.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The following examples are to be construed as merely illustrative andnot a limitation on the scope of the invention in any way. Thespecification refers to the figure in which:

FIG. 1: This shows the solid phase growth of a recombinant C. albicansstrain wherein the MSS4 gene is under the regulation of atetracycline-repressible promoter. The images represent two days growthat 30° C. on SC-U plates with, either no supplement (−DOX), orsupplemented with the tetracycline analogue doxycycline 20 μg/ml (+DOX).The effect of strong induction or tight repression of MSS4 geneexpression on colony formation is observed in the absence and presenceof doxycycline, respectively.

FIG. 2: This shows the liquid phase growth of a recombinant C. albicansstrain wherein the MSS4 gene is under the regulation of atetracycline-repressible promoter. Growth is in YEPD medium at 25° C.with, either no supplement (−DOX, ♦±S.D), or supplemented with thetetracycline analogue doxycycline, 20 μg/ml (+DOX, □±S.D.). The effectof strong induction or tight repression of MSS4 gene expression ongrowth is observed in the absence and presence of doxycycline,respectively.

FIG. 3: This shows the survival rate of mice infected with therecombinant C. albicans strain wherein the MSS4 gene is under theregulation of a tetracycline-repressible promoter. Where indicated, micewere administered with either water (control ♦) or with 2 mg/ml of thetetracycline analogue doxycyline dissolved in 5% sucrose solution (+DOX,□), as drinking water. Under the conditions where the MSS4 gene wasexpressed (control) the C. albicans was able to affect the survival rateof the mice, whereas under the conditions where MSS4 expression wasrepressed (i.e. in the presence of doxycycline) the C. albicans wasunable to impact on the survival rate of the mice.

EXAMPLES Example 1 Expression of MSS4

The MSS4 ORF was cloned into pGex-6P-1 (Pharmacia Biotech) to enableexpression as a 5′ GST fusion protein. Host E. coli used wereTuner(DE3)pLysS (Novagen). E. coli harbouring the expression plasmidwere grown at 37° C. to mid log phase (OD600=0.6) then cooled to 17° C.and induced with IPTG (0.3 mM) for approximately 4 h.

Example 2 Purification of MSS4

E. coli cells from 5L of culture were harvested by centrifugation at6000×g for 10 min. The cell pellets were frozen on −80° C., thawed andresuspended in 150 ml of buffer A (20 mM Hepes (pH7.4), 5 mM DTT, 140 mMNaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.02% (w/v) sodium azide, and aprotease inhibitor cocktail consisting of 1 mM benzamidine, 1 mg.ml-⁻¹each of pepstatin, antipain and leupeptin, 0.2 mM PMSF, Complete (Roche)and general protease inhibitor cocktail (Sigma).

The extract was sonicated in 3×10 s bursts to reduce viscosity. TritonX-100 was added to 1% followed by centrifugation at 75 000×g for 10 min.The supernatant was batch loaded onto 5 ml glutathione Sepharose(Amersham Biosciences) at 4° C. The resin was extensively batch washedwith phosphate buffer containing 0.5M NaCl then with buffer B (20 mMHepes (pH 7.4), 1 mM DTT, 1 mM EDTA, 100 mM NaCl, 10% glycerol, 0.02%sodium azide). The resin was then packed into a disposable PD-10 column.MSS4 was cleaved from GST by incubation of the glutathione Sepharosebeads with 1 column volume of buffer B containing 300U Precisionprotease (Amersham Biosciences) overnight at 4° C. with constanttumbling. MSS4 was recovered by centrifugation of the PD-10 column(500×g) followed by washing of the column with buffer B.

Fractions containing MSS4 were pooled and concentrated to 2 ml using aVivaspin 6 with a 10K cut-off membrane (Vivascience). The protein wasapplied to a Superdex 200 26/10 column linked to an AKTA FPLC system(Amersham Biosciences) and 1 ml fractions were collected. Elutedfractions containing MSS4 were pooled, concentrated and diluted 10-foldwith buffer without NaCl and applied to a Mono Q column (HR5/5) linkedto an AKTA FPLC system (Amersham Biosciences). MSS4 was eluted using alinear 0-500 mM NaCl gradient and MSS4 was eluted at approximately250-350 mM NaCl.

Example 3 MSS4 Assay

The 1-phosphotidylinositol-4-phosphate 5-kinase assay is based on themethod of Desrivieres S, et al, (1998, J. Biol. Chem., 273(25)15787-93). Dried lipids are sonicated into 174, μl of assay buffer (25mM HEPES, pH 7.4, at 25° C., 2 mM MgCl, 0.2 mM EDTA, 1 mM EGTA, 5 mMβ-glycerophosphate, 1 mM dithiothreitol, and 120 mM NaCl) to give afinal concentration in the assay of 100 mMphosphatidylinositol-4-phosphate and 300 mM phosphatidylserine. PurifiedMSS4 is added to the assay (4 μl), and the reactions are started by theaddition of 20 μl of assay buffer containing 0.04 MBq [λ³³P]-ATP and 100μM ATP. Kinase activities are tested at 30° C. for 30 min. The reactionsare stopped by the addition of 750 μl of chloroform: methanol (1:2). Thelipids are then extracted by the addition of 200 μl of 2.4M HCl and 900μl chloroform. The lower phase is washed twice with 1 ml of upper phase(HCl:chloroform:methanol (47:3:48)). The incorporated λ³³P is thenmeasured by transferring 750 μl of the lower phase into a scintillationvial, drying under nitrogen, adding 2 ml of scintillation cocktail, andcounting on a Beckman scintillation counter.

Example 4 MSS4 as an Essential Gene Product

4.1 Construction of the Tetracycline-Regulatable Expression System

C. albicans CAI8 was used as the parental strain for all manipulations.The parental strain was constructed to constitutively express acodon-optimised tetracycline transactivator, consisting of TetR fused tothe viral VP16 transcriptional activation domain (Gari E, et al, 1997,Yeast, 13:837-848), from the chromosomal enolase promoter (Mason A B, etal, 1993, J. Bacteriol., 175: 2632-2639). One copy of the target gene,MSS4, was disrupted in the transactivator expressing strain using thestandard URA-blaster method (Fonzi W A & Irwin M Y, 1993, Genetics,134:717-728). The promoter region of the other MSS4 allele was thenreplaced with the minimal promoter element containing the tetracyclineoperator sequence tetO. Both in vivo and in vitro this system enablesstrong induction and tight repression of MSS4 gene expression in theabsence and presence, respectively, of the tetracycline analoguedoxycycline.

4.2 In Vitro Validation Experiments

The essential nature of MSS4 was determined by assessing the growth andviability of the C. albicans strain modified to include atetracycline-regulatable MSS4-expression system as described above(section 4.1). A single fresh colony (grown at 30° C. in rich medium inthe absence of tetracycline) was used to streak fresh plates containingsynthetic complete medium minus uracil (SC-U) (Qbiogene), plus 2% agar,plus or minus 20 μg/ml doxycycline as indicated. Growth was scored after2 days at 30° C. (FIG. 1). Very few colonies were observed under theconditions where MSS4 expression was repressed (i.e. in the presence ofdoxycycline).

Growth of the tetracycline-regulatable MSS4-expression system C.albicans strain in liquid YEPD medium was also assessed. The inoculumwas a 1:100 dilution of an overnight culture adjusted with PBS to anoptical density at 600 nm=1 and stored at 4° C. Growth, at 25° C, plusor minus 20 μg/ml doxycycline, was measured at 30 min intervals over a43 h time period or until the growth had noticeably reached a plateau.Growth curves were recorded in 96 well plates in a Wallac plate reader(600 nm, 25° C. heated stage, 2 mm orbital shaking pattern) (FIG. 2).Again, significantly reduced growth was observed under the conditionswhere MSS4 expression was repressed (i.e. in the presence ofdoxycycline).

Example 5

MSS4 as an Essential Gene Product: In Vivo Validation Experiment—MurineModel of Systemic Infection with C. albicans Conditional Mutants

Several reproducible animal models have been described including thoseof rat vaginal and oral Candidiasis (Calderone R A & Braun P C, 1991,Microbiol. Rev., 55:1-20). However, the most commonly used model is themurine model of hematogenously inoculated, disseminated Candidiasis(Ghannoum M A, et al, 1995, Infect. Immun., 63:4528-4530). In the murinedisseminated model, a single dose of organism is inoculated via the tailvein. The end points for this model are survival of animals and tissuecounts of C. albicans (generally the kidneys).

To further validate the essential nature of the MSS4 gene, theconditional mutants of C. albicans strains (as described in Example 4,section 4.1), wherein the MSS4 gene is under the control of atetracycline repressible promoter, were tested to determine whether theywere attenuated in an immuno-competent murine model of infection(Ghannoum et al, 1995) as follows:

Initially the organisms were grown in the absence of DOX, since underthese conditions they would express the MSS4 gene. These organisms werethen used to inoculate two groups of 12 of mice. One group was treatedwith DOX, in which expression of the MSS4 gene was repressed, and thesecond group of mice was treated with water (control) wherein the genecontinued to be expressed. The mice used were single sex BALB/c mice,Harlan, 4 weeks old and weighing between 19-22 g.

Infective doses of C. albicans were injected into the tail vein. Theinocula were from saline-washed fresh stationary phase cultures grown inNGY medium [0.1% neopeptone, 0.4% glucose, 0.1% yeast extract] for 18-24h at 30° C. Yeasts grown in this way have a viable count of 2×10⁷CFU/ml±0.3×10⁷ CFU/ml and can easily be adjusted to the desiredconcentration with saline. The concentration was checked byspectrophotometry and verified by viable counts. The volume injected wasthe same across the doses. An infective dose of 1×10⁶ CFU/mouse wasused. This infective dose has previously been shown to give a meansurvival time of 5-7 days in BALB/c mice.

Animals were fed food and water ad libitum throughout the course ofexperiment. In the DOX-treated group (+DOX), mice were administered withDOX (2 mg/ml) dissolved in 5% sucrose solution as drinking water from 2days before the inoculation of C. albicans cells. The mice are known todrink approximately 5 ml of sucrose solution every day. Under thisregimen, the concentrations of DOX in serum, liver, and kidney aremaintained at more than 2 mg/ml of serum, 8 mg/g of liver, and 10 mg/gof kidney, respectively (Nakayama, H., et al., 1998, Microbiology144:2407-2415.) Percent survival was followed over 28 days with dailybody weight monitoring. Differences between the effects of C. albicanswith the MSS4 gene active (−DOX) or repressed (+DOX) in vivo weremonitored by mouse survival (see FIG. 3), kidney burdens of viablefungi, and changes in body weight relative to baseline.

Kidney counts (colony forming unit counts per gram of kidney tissue) forthe (+DOX) group were significantly reduced compared to the controlgroup (−DOX) and these mice also maintained their weight throughout thecourse of the study.

1. A method of screening or testing for candidate anti-fungal compoundsthat impair 1-phosphotidylinositol-4-phosphate 5-kinase enzyme (MSS4)function, comprising: a) providing fungal MSS4; b) providing one or morecandidate compounds; c) contacting said MSS4 with said one or morecandidate compounds; and d) determining the interaction of the candidatecompound with said MSS4.
 2. A method according to claim 1 wherein theMSS4 comprises a fragment, a function-conservative variant, an activefragment or a fusion protein of MSS4.
 3. A method according to claim 1,wherein the fungal MSS4 is from fungus of Candida or Aspergillusspecies.
 4. A modified eukaryotic cell(s) wherein the cell(s) expressesfungal MSS4 under the control of a heterologous promoter.
 5. The cellaccording to claim 4 which is a C. albicans cell.
 6. The cell accordingto claim 4, wherein the MSS4 is homologous.
 7. The cell according toclaim 4, wherein the MSS4 comprises a fragment, a function-conservativevariant, an active fragment or a fusion protein of MSS4.
 8. A method ofscreening or testing for candidate anti-fungal compounds that impair1-phosphotidylinositol-4-phosphate 5-kinase enzyme (MSS4) function,comprising: a) providing fungal MSS4 in a eukaryotic cell(s) as definedin claim 4; b) providing one or more candidate compounds; c) contactingsaid eukaryotic cell(s) with said one or more candidate compounds; andd) determining the interaction of the candidate compound with said MSS4by assessing the effect on growth or viability of said cells.
 9. Acompound identified by the method of claim 1, which impairs MSS4function for use as an antifungal compound.
 10. A pharmaceuticalcomposition comprising a MSS4 inhibitor and a pharmaceuticallyacceptable carrier.
 11. Candida or Aspergillus MSS4 as a specific targetfor antifungal compounds.
 12. (canceled)
 13. (canceled)
 14. The methodaccording to claim 18 wherein the fungal infection is a topical, mucosalor systemic fungal infection.
 15. The method according to claim 14wherein the topical or mucosal fungal infection is caused by species ofCandida or the systemic fungal infection is caused by species of Candidaor Aspergillus.
 16. The method according to claim 18 wherein saidcompound impairs fungal MSS4 function to a greater extent than host MSS4function.
 17. A compound identified by the method of claim 8, whichimpairs MSS4 function for use as an antifungal compound.
 18. A methodfor the treatment or prevention of fungal infections in a host, whichcomprises administering to the host a therapeutically orprophylactically effective amount of a MSS4 inhibitor.
 19. A method forthe treatment or prevention of fungal infections in a subject who isimmunosuppressed, which comprises the step of administering to thesubject a therapeurtically or prophylactically effective amount of aMSS4 inhibitor.
 20. The method according to claim 19 wherein the fungalinfection is a topical, mucosal or systemic fungal infection.
 21. Themethod according to claim 19 wherein the topical or mucosal fungalinfection is caused by species of Candida or the systemic fungalinfection is caused by species of Candida or Aspergillus.
 22. The methodaccording to claim 19 wherein said compound impairs fungal MSS4 functionto a greater extent than host MSS4 function.